**2.4 The effect of glycerol in synthetic wine media on the activity of glucose isomerase**

The synthetic wine media were prepared with 1% w/v fructose, 13% v/v ethanol, and different glycerol contents. The flasks were incubated at 60°C for 47 hours. In order to simulate the wine environment, the pH was adjusted to 3.3 with 1:1 acetic acid solution.

### **2.5 The effect of sulfite content on the activity of glucose isomerase**

The synthetic wine medium contained 1% w/v fructose as a substrate with different sulfite amounts. The flasks were incubated at 60°C and 150 rpm for 14.5 hours. The pH values of solutions were adjusted to 3.5 with 1:1 acetic acid solution.

#### **2.6 The effect of tannins in red wine on the activity of glucose isomerase**

In this experiment, 1 g of fructose was added to a 100 mL wine medium. Samples were incubated in flasks at 60°C and 150 rpm for almost 70 hours. The pH values of solutions were 3.37, 3.05, and 3.5 for Doluca Mistik Red, Turkey, 2016 (alcohol: 14.0% v/v) (w1), Doluca Mistik White, Turkey, 2016 (alcohol: 13.5% v/v) (w2), and Frontera, Chile, 2015 (alcohol: 12.5% v/v) (w3), respectively.

#### **2.7 The effect of fermentation components on the activity of glucose isomerase**

To obtain synthetic wine media with grape juice, 5% w/v fructose, 13% v/v ethanol, and 0.8% v/v glycerol were mixed with grape juice. Two types of grape juices were used during the experiments. The industrial white and red grape juices that were obtained from the market were Kavaklıdere brand. Also, the homemade grape juice was tested and used during the experiments. Samples were incubated in flasks at 60°C and 150 rpm. Since grape juice already contained Mg+2 [14], the enzyme did not require an additional activator. The pH of homemade grape juice was 3.65 and 3.88 for synthetic wine media with homemade grape juice.

#### **2.8 The effect of calcium content on the activity of glucose isomerase**

In this experiment, 1% w/v fructose was used as a substrate, and 13% v/v ethanol and 0.8% v/v glycerol were added to provide synthetic wine environment. The pH values of the media were adjusted to 3.6 with 1:1 acetic acid solution. Since 100 mL red wine contains 8 mg calcium, the same amount of calcium must be present in the synthetic wine medium to provide the same conditions. Therefore, 0.015 g Ca (OH)2 was added to 100 mL synthetic wine medium. The flasks were incubated at 60°C and 150 rpm for almost 65 hours. Another experiment was conducted by considering the ion retention capacity of EDTA to hold the calcium in homemade red wine media with alcohol content of 14% v/v. Different concentrations of EDTA were added to red wine media containing 1% w/v fructose as a substrate. The flasks were incubated at 60°C and 150 rpm. The pH of solutions with 0.1, 0.2, 0.6, 1, and 2% w/v EDTA were 3.46, 3.43, 3.53, 3.53, and 3.6, respectively. For the cation exchanger experiment, to test the enzyme activity in red wine media containing little amounts of calcium, 1% w/v fructose was added to sample numbers 0, 4, 6, and 10. The numbers of samples were named from 0 to 10. The number of 0 was the original wine sample, and other numbers were wine samples that were eluted through the resin at a rate of 40 mL per sample. While sample 0 contained almost 13 ppm calcium in it, other samples contained much less amounts of calcium with respect to sample 0. The pH values of samples were 3.6, and they were incubated at 60°C and 150 rpm for almost 80 hours.

#### **2.9 The activity of glucose isomerase in diluted wine media**

In the first experiment, homemade red wines were used containing 1% w/v fructose (added after dilution) as a substrate. Wines were diluted with distilled water at six different concentrations: 90, 70, 50, 30, 20, and 5% v/v and also 100% v/v red wine as a control. The prepared solutions were incubated at 60°C and 150 rpm for 42 hours. The pH values of solutions were 3.15, 3.15, 3.19, 3.3, 3.53, 3.59, and 4.45 for wines at concentrations of 100, 90, 70, 50, 30, 20and 5% v/v, respectively. Also, different brands and types of wines; w1, w2, and w3; at different concentrations, 100, 10, and 5% v/v, were used in another experiment. The samples containing 1% w/v fructose as a substrate were incubated at 60°C and 150 rpm for 27 hours. The pH values of solutions were 3.5, 3.45, and 3.03 for 100% v/v w3, w1, and w2, respectively.

#### **2.10 The effect of tartaric acid on the activity of glucose isomerase**

Synthetic wine environments with 1% w/v fructose and 0.3% w/v tartaric acid were prepared at pH values of 3.55 and 6.33 adjusted with 5 M NaOH and 24% w/v KOH solutions, respectively. Samples were incubated at 60°C and 150 rpm. For experiments, samples numbered as 0, 2, 5, 8, 9, 10, and 12 were chosen with the addition of 1% w/v fructose. The pH values of solutions, 0, 2, 5, 8, 9, 10, and 12, after fructose addition were 3.36, 9.85, 6.7, 4.8, 4.08, 3.83, and 3.66, respectively. Samples from 0 to 13 were passed through anion exchanger resin. The first 9 samples passed through an anion exchanger had higher pH values than the original sample. In the original sample, number 0, the tartaric acid content was equal to 0.248% w/v. Other samples from 1 to 13 did not contain any tartaric acid. The samples were incubated at 60°C and 150 rpm.

*Determining Glucose Isomerase Activity in Different Wine Environments to Prevent Sluggish… DOI: http://dx.doi.org/10.5772/intechopen.100499*
